A deep understanding of the genetic mechanisms of musculoskeletal development and disease requires study of genes that are frequently pleiotropic and/or highly redundant. Currently, the common strategy to study such complex genetic traits in mice is to combine multiple homozygous conditional (floxed) alleles with a tissue-specific and/or temporally inducible Cre recombinase transgene. However, such breeding strategies to understand three or more loci require an extraordinary investment of money, time, and mice to obtain just a few animals of the desired genotype, often limiting enquiry to a particular tissue and developmental stage. Here, we propose an innovative approach in mice using just three hemizygous transgenes to conditionally induce multi-gene, bi-allelic, loss-of-function mutations to study complex genetic control of development and maintenance of the limb skeleton. We previously demonstrated highly efficient CRISPR/Cas9 mutagenesis from genetically encoded elements that included the Rosa26-LoxStopLox-Cas9 transgene. Crossing these mice with a tissue-specific and/or temporally-inducible Cre transgenic line provides an opportunity to control the timing and/or location of CRISPR/Cas9 activity. The third transgene required for this proposed system is a `polycistronic tRNA-gRNA' (PTG) array of CRISPR guide RNAs interspersed with tRNA sequences. These transcripts recruit endogenous RNases to cleave apart gRNAs, and processed gRNAs can complex with Cas9 protein to induce sequence-targeted double strand breaks in DNA. PTG arrays have been effective in rice, Drosophila, zebrafish, and cultured human cells, but they remain untested in mice. Using this strategy, we plan to engineer and validate two models of skeletal development. In Aim 1, we will engineer a Smad1, Smad5, and Smad8 conditional knockout strategy to target loss of BMP/GDF signaling at a critical bottleneck. This will be a valuable model to thoroughly assess pathway function during limb patterning, digit condensation and interdigital cell death, chondrocyte specification and maintenance, and osteoblast specification and maintenance. In Aim 2, we will engineer an aquaglyceroporin (AQP3, AQP7, and AQP9) conditional knockout strategy. This model will allow us to test the hypothesis that these membrane channels that increase plasma membrane permeability to water and small uncharged osmolytes are necessary for growth plate hypertrophic chondrocyte swelling and maintenance of articular cartilage. Since the purpose of this R21 proposal is the engineering and validation of new technology and animal models, our primary emphasis will be to determine the efficiency and reproducibility of such strategies and to establish a set of guidelines for future implementation. Successful outcomes of this work will provide a wealth of opportunities to understand the importance of these two complex genetic systems, and the strategy has broad potential to accelerate research and reduce the costs associated with mouse models of development and disease.